DESCRIPTION: Highly active antiretroviral therapies (HAART) is incapable of fully eradicating virus within an adult patient, due to the persistence of latently infected resting CD4+ T lymphocytes and infected macrophages and monocytes, which may produce low levels of viremia without dying. Recently, there has been a growing interest in using gene therapy as an alternative to HAART, particularly with the recent breakthrough of the Berlin patient, who was functionally cured of HIV after receiving an allogeneic bone marrow transplant with CCR5-defective hematopoietic stem cells. Indeed, we have conducted the only clinical trial to date to test an anti-HIV RNAi therapeutic, which required the ex vivo delivery of a lentiviral vector in autologous CD34+ hematopoietic stem cells for patients with AIDS-related lymphoma. However, for RNAi-based therapeutics to become available for the vast majority of HIV patients, alternative systemic delivery methods must be developed. In this project, we will test three promising methods for delivering anti-HIV siRNAs: an anti-gp120 RNA aptamer-siRNA conjugate for targeted delivery into cells actively infected with HIV; an anti-CD4 RNA aptamer-siRNA conjugate for targeted delivery into CD4+ T cells and macrophages; and a cationic PAMAM dendrimer-siRNA nanoparticle for systemic delivery. For Aim 1, we will elucidate the molecular interactions between the anti-gp120 aptamer and HIV gp120 protein using surface plasmon resonance, X-ray crystallography, and electron microscopy. In Aim2 of this project, we will use positron emission tomography and near-infrared fluorescence imaging to measure the dose- dependent biodistribution and whole-body clearance of our RNA-based therapeutics in a humanized mouse model. In Aim 3, we will conduct a series of in vivo experiments in HIV-infected humanized mice to test the efficacy of individual and combination RNA-based therapies. These experiments will test siRNAs that target endogenous genes required for HIV infection (prophylactic and entry inhibitor siRNAs), mechanisms required for the persistence of latency (anti-latency siRNAs), and genes required for cell survival (cytotoxic siRNAs). Hence, we will test a comprehensive, RNA-based shock and kill strategy that aims to purge the latent reservoir, eliminate the cells actively infected with HIV, and protect cells from new infection. In addition to standard assays for detecting HIV viremia, we will use droplet digital PCR to measure the levels of integrated provirus in treated and non-treated animals. In summary, this project represents an innovative approach for suppressing and potentially eradicating HIV infection. Moreover, the aims of this pre-clinical study will establish an important benchmark for evaluating RNAi- based therapy as a treatment for HIV.